Large-scale mutational analysis and accelerated evolution of genes of microorganisms or other cells has been aided by in vitro mutagenesis in combination with epichromosomal expression in model organisms followed by analysis of these site-specific changes in the model systems.
However, techniques for random mutagenesis of extended genomic regions in their native genetic context suffer from serious limitations. Currently there are four different ways to elevate the in vivo mutation rate in extended regions of the cellular DNA.
The first type of protocols takes advantage of chemical mutagens, ultraviolet light or hypermutator strains and introduces mutations everywhere into the cellular DNA in an untargeted way. These methodologies lead to the accumulation of numerous undesired, off-target modifications outside a PTR posing detrimental side effects for the organism to be evolved.
The second type of mutagenesis protocols targets mutations to a PTR, but introduces them in vitro using error-prone PCR or site-saturation mutagenesis and recombines the resulting genetic repertoire into cellular DNA subsequently using CRISPR-Cas9-mediated engineering. Basically, these methods demand prior genomic modification of the host organism or time-consuming mutagenic donor template generation.
The third type of mutagenesis protocol introduces a double strand DNA break into cellular DNA and harnesses the natural increase in the mutation rate (up to 800 fold) during the break repair. With this technique there is very little control over the type and location of mutations around a break.
The fourth type of protocols applies targeted, enzymatically-induced DNA damage to achieve mutagenesis on a short PTR (<100 consecutive nucleotides).
These methods lack the ability to provide bias-free and off-target-effect free mutagenesis on extended genomic loci.
Recent progress has been made in single stranded DNA (ssDNA) recombineering, broadening the toolset for in vivo bacterial mutagenesis techniques.
Most promising among these is MAGE (Multiplex Automated Genome Engineering) which enables the efficient recombination of ssDNA oligonucleotides (oligonucleotides or oligos) into the cellular DNA (inducing in vivo synthesis of the cellular DNA through hybridizing the oligonucleotides to the PTR) and has been applied to introduce vast combinatorial mutational diversity (Wang et al. 2009). With good control over the mutational spectra and the PTR, MAGE has allowed the specific in vivo mutagenesis of short sequences such as ribosome binding sites or directed evolution of certain residues in protein coding sequences (Diner and Hayes 2009; Amiram et al. 2015). In these experiments, however, the maximum number of mutagenized neighboring nucleobase positions was always less than 30 residues. MAGE has never been demonstrated for longer continuous sequences. This is due to the fact that MAGE's efficiency is largely dependent on oligo interaction with the target sequence (Wang and Church 2011; Gallagher et al. 2014), Specifically, the efficiency of oligonucleotide integration decreases with the increasing number of mutagenized positions in the PTR, putting a practical limit to the number of positions that can be randomized with a single oligo. Additionally, efficient integration of the oligo requires strict sequence identities to the PTR at the extremities of the oligos. Thus, the PTR undergoing mutagenesis must be positioned to the center of single ssDNA oligonucleotides. The net result of these constraints is that diversification of every positions of a PTR longer than ˜30 bps is not feasible using a conventional oligo design strategy.
Norwald et al. propose a tool for multiplexed DNA synthesis and homologous recombination to construct rational libraries (Nordwald et al. 2013). Recombineering is described as a method for producing genetic diversity and creating sequence-to-activity mapping libraries for protein engineering. In this method, each single amino acid substitution is introduced by a separate, rationally designed ssDNA oligo, limiting the number of possible nucleobase alterations. Reference is made to a multi-step strategy using MAGE for parallel combinatorial optimization of proteins.
U.S. Pat. No. 6,391,640 B1 discloses the evolution of genes or metabolic pathways by recursive sequence recombination (U.S. Pat. No. 6,391,640 B1: Methods and compositions for cellular and metabolic engineering). Initial substrates for recombination are cloned into a plasmid vector. A diversity of substrates may be used. Such diversity can be produced by mutagenesis.
WO 00/42561 A2 discloses oligo mediated nucleic acid recombination for in vitro DNA shuffling, thereby producing a diversity of nucleic acids (WO 00/42561 A2: Enzymes, pathways and organisms for making a polymerizable monomer by whole cell bioprocess).
Coussement et al. describe a one-step DNA assembly for combinatorial metabolic engineering. Two promoter libraries were simultaneously introduced in front of two target genes (Coussement et al. 2014).
Daiguan Yu et al. describe recombineering in E. coli using overlapping DNA oligonucleotides (Yu et al. 2003). Multiple overlapping oligos are described to be useful for making complex constrcuts in vivo without the need for restriction enzymes or DNA ligase.
DiCarlo et al. describe yeast oligo-mediated genome engineering for allelic replacement in yeast for short sequences (DiCarlo et al. 2013).
WO2014/102688A1 discloses a donor matrix to perform homologous recombination in cells wherein said matrix is made of single stranded oligonucleotides that partially hybridize with each other over a complementary sequence. According to an example, the matrix is used for targeted genetic modification by homologous recombination, introducing an exogenous sequence into a genomic locus.
WO02/14495A2 discloses enhanced homologous recombination mediated by lambda recombination proteins. The DNA used in the method is a single oligonucleotide sequence, or may be two or more overlapping sequences.
There is a need for new cost effective methods for in vivo cellular mutagenesis which could cover a large PTR.